Developmental Biology 205, 111–128 (1999)
Article ID dbio.1998.9109, available online at http://www.idealibrary.com on
On the Control of Oocyte Meiotic Maturation and
Ovulation in Caenorhabditis elegans
James McCarter, Bart Bartlett, Thanh Dang, and Tim Schedl 1
Department of Genetics, Washington University School of Medicine, St. Louis, Missouri 63110
Prior to fertilization, oocytes undergo meiotic maturation (cell cycle progression) and ovulation (expulsion from the ovary).
To begin the study of these processes in Caenorhabditis elegans, we have defined a time line of germline and somatic events
by video microscopy. As the oocyte matures, its nuclear envelope breaks down and its cell cortex rearranges. Immediately
thereafter, the oocyte is ovulated by increasing contraction of the myoepithelial gonadal sheath and relaxation of the distal
spermatheca. By systematically altering the germ cell contents of the hermaphrodite using mutant strains, we have
uncovered evidence of four cell– cell interactions that regulate maturation and ovulation. (1) Both spermatids and
spermatozoa induce oocyte maturation. In animals with a feminized germline, maturation is inhibited and oocytes arrest
in diakinesis. The introduction of sperm by mating restores maturation. (2) Sperm also directly promote sheath contraction.
In animals with a feminized or tumorous germline, contractions are infrequent, whereas in animals with a masculinized
germline or with sperm introduced by mating, contractions are frequent. (3 and 4) The maturing oocyte both induces
spermathecal dilation and modulates sheath contractions at ovulation; dilation of the distal spermatheca and sharp
increases in sheath contraction rates are only observed in the presence of a maturing oocyte. © 1999 Academic Press
Key Words: oocyte maturation; meiosis; ovulation; cell– cell interaction; sperm; myoepithelial contraction and relaxation; Caenorhabditis elegans.
INTRODUCTION
Oocyte maturation is a cell cycle event which releases
the oocyte from meiotic prophase (similar to a G2 to M
phase transition) and allows progression through the meiotic divisions and fertilization (Masui and Clarke, 1979;
Wickramasinghe and Albertini, 1993; Downs, 1995).
Nuclear envelope breakdown (NEBD or germinal vesicle
breakdown) is its most characteristic feature. Maturation
is preceded by oocyte development (oogenesis), and is
rapidly followed by ovulation, releasing the oocyte from
the ovary.
Stimuli which can induce oocyte maturation are known
in some animals. Maturation is induced by application of
1-methyladenine in starfish (Kanatani et al., 1969) and
serotonin in clams (Krantic et al., 1991). In vertebrates,
increased release of gonadotropins by the pituitary signals
the somatic cells of the follicle for maturation. In some
1
To whom correspondence should be addressed at Department
of Genetics, Washington University School of Medicine, Box 8232,
4566 Scott Ave., St. Louis, MO 63110. Fax: (314) 362-7855. E-mail:
[email protected].
0012-1606/99 $30.00
Copyright © 1999 by Academic Press
All rights of reproduction in any form reserved.
vertebrates, such as amphibians, progesterone is then produced by follicle cells and apparently signals the oocyte to
mature (Masui, 1967). In mammals, the mechanism by
which the follicular granulosa cells cause oocyte maturation is unresolved, but may involve overcoming inhibition
by cAMP (Eppig, 1993). In the genetic model systems
Caenorhabditis elegans and Drosophila the molecular signals for oocyte maturation are unknown.
The study of Xenopus oocyte maturation (Masui and
Markert, 1971) led to the isolation of maturation promoting
factor (MPF), a cyclin B/Cdc2 kinase complex which acts
within the oocyte as the crucial regulator of maturation
(Lohka et al., 1988; Maller et al., 1989). Activated MPF
phosphorylates targets responsible for NEBD and other
events which mark the transition from meiotic prophase to
metaphase. Cyclins and cyclin-dependent kinases are evolutionarily conserved and play crucial roles in cell cycle
regulation of both mitosis and meiosis in organisms from
yeast to humans (Nurse, 1990). Studies in Xenopus and
mouse also indicate a role for the kinase Mos and the MAP
kinase cascade in oocyte maturation and/or the transition
from meiosis I to meiosis II (Sagata et al., 1989; Kosako et
al., 1994; Verlhac et al., 1994).
111
112
McCarter et al.
TABLE 1
Strains Used in This Study
Linkage group a
LG I
LG II
LG IV
LG V
a
b
Strain
Reference b
N2 (wild-type reference strain, Bristol)
BS390 fog-1(q180)unc-13(e51)/hT2
CB51 unc-13(e51) and CB1091 unc-13(e1091)
BS1 spe-4(q347)/unc-109(n499sd)
BS146 gld-1(q266)/hT2
BS406 unc-13(e51)gld-1(q485)fog-3(q443)/hT2
BS273 unc-13(e51)gld-1(oz10)/unc-55(e402)
BA76 fer-1(hc13); him-5(e1490)
SL154 fer-1(b232ts); him-5(e1490)
SL170 fer-1(hc47)dpy-5(e61)/hT2; him-5(e1490)
BS3075 unc-13(e51)fog-3(q443)hT2
BS196 fog-3(q443)/lin-10(e1439)unc-29(e193)
BS3158 glp-4(bn2); him-5(e1490)
CB120 unc-4(e120)
BA3 fer-3(hc3)
CB138 unc-24(e138)
BS3046 unc-24(e138)fem-3(e1996)/DnT1
BS3147 fem-3(e1996)/DnT1
JK816fem-3 (q20gf)
JK574fog-2(q71)
—
Barton and Kimble, 1990
—
L’Hernault et al., 1988
Francis et al., 1995
Francis et al., 1995
Francis et al., 1995
Ward and Miwa, 1978
S. L’Hernault, personal communication
S. L’Hernault, personal communication
Ellis and Kimble, 1995
Ellis and Kimble, 1995
Beanan and Strome, 1992
—
Argon and Ward, 1980
—
Hodgkin, 1986
Hodgkin, 1986
Barton et al., 1987
Schedl and Kimble, 1988
Strains are organized by the linkage group of the gene of interest to this study.
References for the genes of interest to this study are provided. References for all other genes are provided in Hodgkin (1997).
Ovulation is the physical process whereby the oocyte is
released from the ovary and becomes available to sperm for
fertilization. Ovulation is temporally coupled to maturation in most metazoans, although its features are less
conserved. For instance, vertebrate ovulation requires the
rupture of the ovarian wall by the follicle in a proteolytic
cascade comparable to the inflammatory response (Espey
and Lipner, 1994), whereas in C. elegans and Drosophila
myoepithelial sheath cells in the ovary contract at ovulation to move the oocyte out through a passage which
directly connects to the spermatheca and uterus (King,
1970; Hirsh et al., 1976). In C. elegans the distal spermathecal cells form a stricture at the proximal end of the ovary (or
gonad arm) and only dilate to let the oocyte pass at
ovulation.
C. elegans is an excellent model for addressing the
regulation of oocyte maturation and ovulation. The hermaphrodite has a female soma with a germline that makes
sperm first and then produces oocytes in both gonad arms.
Full-grown oocytes mature and are ovulated and fertilized
in a single file and assembly-line-like fashion. In addition to
its utility as a genetic system (Hodgkin et al., 1988) and
defined cellular lineages (Kimble and Hirsh, 1979), the
worm is transparent and can be anesthetized (Kirby et al.,
1990) to allow video recordings of late oogenesis, oocyte
maturation, and ovulation. Defective ovulation has already
been examined by several groups; disruption of the gonadal
sheath’s contractile function by laser ablation (McCarter et
al., 1997) or mutation (Myers et al., 1996; Iwasaki et al.,
1996; Rose et al., 1997) leads to failure of ovulation and
sterility with mature oocytes trapped in the gonad arm.
Using time-lapse microscopy with wild-type worms, we
have defined a time line of landmark morphological events
during oocyte growth, maturation, and ovulation in C.
elegans. By manipulating the presence or absence of oocytes
and sperm in the worm using mutant strains and matings,
we have obtained evidence of four cell– cell interactions
that regulate maturation and ovulation in this species.
MATERIALS AND METHODS
Nematode Strains, General Methods, and
Terminology
Methods for C. elegans culture and manipulation were as described (Sulston and Hodgkin, 1988). Strains were grown at 15 or
20°C. Observations and video recordings were made at room
temperature (20 –23°C) unless otherwise noted. Strains used are
listed in Table 1.
The following definitions used here are based on the events that
can be visualized by Nomarski microscopy and the reproductive
biology of C. elegans (also see Results and Discussion). Meiotic
maturation: the transition from diakinesis of prophase I to metaphase of meiosis I. Events include nuclear envelope breakdown
(NEBD), which begins in the gonad arm prior to ovulation and
finishes in the spermatheca, and oocyte cortical rearrangement,
which occurs in the gonad arm prior to ovulation. Oocyte: female
germ cell up to the time of fertilization. Egg: zygote after the point
of fertilization, which is normally followed by egg shell formation.
Copyright © 1999 by Academic Press. All rights of reproduction in any form reserved.
113
Oocyte Maturation in C. elegans
This definition of egg/embryo includes the possibility that C.
elegans maternal effect embryonic lethal mutations (Kemphues
and Strome, 1997) may exist where eggs are produced (fertilization
and egg shell formation) and yet events of meiotic maturation (i.e.,
NEBD) are defective.
Nomarski and Fluorescence Microscopy
Observations of living animals by Nomarski (DIC) microscopy
were as described (Sulston and Hodgkin, 1988) using a Bmax-60F
(Olympus, Inc.) microscope. For fluorescence microscopy, nematode gonads were dissected, fixed, and stained as described (Francis
et al., 1995). Images were collected using an Optronics DEI-470
cooled CCD camera, transferred to a Power Macintosh 7100
(Apple, Inc.) running NIH Image 1.58 (Wayne Rasband, NIH),
assembled with Photoshop 3.0 (Adobe, Inc.), and printed on a
Phaser 440 dye-sublimation printer (Tektronix, Inc.).
Nomarski Time-Lapse Microscopy
For time-lapse observations, worms were anesthetized for 30 –45
min in a solution of M9 with 0.1% tricaine and 0.01% tetramisole
(Sigma, Inc.) before viewing (Kirby et al., 1990; McCarter et al., 1997).
Tricaine/tetramisole blocks body wall movement, pharyngeal pumping, and egg laying. Events of late oogenesis, oocyte maturation,
ovulation, and fertilization all continue undisturbed for the first 4 –5
oocytes in the arm, while new oocytes are not formed at the loop of
the gonad arm (possibly because nutrient availability from the intestine diminishes). Worms can be recovered from an anesthetic exposure of up to 4 h. Animals were mounted on an Axioskop (Zeiss, Inc.)
microscope and viewed with low light using the 403 or 633 lens. To
prevent heating, an infrared filter was added to the light path. The
microscope was connected to a XC-75 CCD video camera (Sony, Inc.)
or a DEI-470 cooled CCD video camera (Optronics, Inc.). Time-lapse
recordings were made at 1/12 real time.
Quantitation of Maturation Rates, Oocyte Volume,
Oocyte Nuclear Volume, and Sheath Activity
Maturation is the rate-limiting step in the production of fertilized eggs in adult hermaphrodites (see Results). The rate at which
oocytes undergo maturation was calculated by measuring total
embryo production in adult hermaphrodite populations and by
direct observation of individual animals with video microscopy.
For both wild-type and mutant strains the two methods were in
excellent agreement. To measure total embryo production in a
population, animals were synchronized as described (McCarter et
al., 1997). Animals were mounted in M9 for Nomarski microscopy
and the total number of embryos in the uterus of each animal was
determined. Animals were transferred to fresh plates with Escherichia coli for several hours. Upon completion of the time interval,
embryos in the uterus and laid on the plate were counted. The
oocyte maturation/ovulation rate per gonad arm per hour 5 (number of embryos at end of interval 2 number of embryos at beginning
of interval)/(2 3 population size). The average population size used
per trial was 19 and average time interval was 3.5 h.
Oocyte and oocyte nuclear volume were calculated by direct
measurement of oocyte width and length, and nuclear diameter on
the monitor during video production. Sizes were calibrated to
convert cm on the monitor to true micrometer values. We assumed
the oocyte nucleus to approximate a true sphere so that volume 5
4/3 3 p 3 (diameter/2) 3. We assumed the oocyte cell to approxi-
mate a true cylinder so that volume 5 p 3 length 3 (width/2) 2.
Sheath contractile activity was quantitated by replaying recorded
videos and visually counting the number of contractions occurring
in the myoepithelium over 1-min intervals. Contractions were
counted twice and averaged.
RESULTS
C. elegans Oocytes Are Produced in an AssemblyLine-like Process That Reaches a Steady-State Rate
in the Adult Hermaphrodite
The C. elegans hermaphrodite reproductive system is
made up of two gonad arms, each connected by a spermatheca to the common uterus; gametogenesis occurs in
the arms and fertilization in the spermatheca. Oocytes are
produced and leave the gonad in a single-file assembly-linelike process so that following maturation, ovulation, and
fertilization of the most proximal (first) oocyte, the second
oocyte takes the most proximal position, and the third
takes the position of the second, etc. (Fig. 1).
The production of fertilized eggs includes the processes of
oocyte growth and development, maturation, ovulation,
and fertilization, and theoretically any one of these steps
could be rate limiting. Examination of oocytes by timelapse video Nomarski microscopy reveals that in adult
hermaphrodites, oocyte maturation is the rate-limiting step
with ovulation and fertilization immediately following
each maturation. Oocytes mature at a rate of 2.7 6 1.3 per
gonad arm per hour, or one every ;23 min (range 11 to 42
min, n 5 19 oocytes). This rate agrees closely with the rate
of embryo production determined for unanesthetized adult
animals on plates. In young adult hermaphrodites producing their first several oocytes, oocyte growth and development prior to maturation is rate limiting with only 1.3 6
0.3 oocytes produced per gonad arm per hour, or one every
;47 min (range 34 to 83 min, n 5 17 oocytes). Young adults
have only ;5 or fewer oocytes in the proximal arm, whereas
mid-stage adults can have a backlog of ;10 oocytes filling
the proximal arm and loop. Further, oocytes in young adults
are still increasing in size as they near the most proximal
position, whereas oocytes in adults reach their full size
earlier in their progression through the proximal arm.
Oocyte Development, Meiotic Maturation, and
Ovulation Are Defined by Reproducible Landmark
Events
The events of late oogenesis, meiotic maturation, and
ovulation in the adult hermaphrodite are depicted for one
oocyte along a time line in Fig. 2 and presented as individual micrographs in Fig. 3. Completion of ovulation with
the closure of the distal spermatheca is defined as the
reference zero time point and corresponds closely to the
time of fertilization. (Prior events receive negative time
values.) Table 2 presents the average time for each event (6
standard deviation) and additional features of the events. A
Copyright © 1999 by Academic Press. All rights of reproduction in any form reserved.
114
McCarter et al.
FIG. 1. Schematic of a C. elegans adult hermaphrodite gonad arm. Each of the two gonad arms is a U-shaped tube that generates male and
female gametes. Germ cells proliferate distally and then enter and progress through meiotic prophase as they move proximally. The first ;40
germ cells to initiate meiotic development undergo spermatogenesis in the proximal region of each gonad arm and complete the divisions of
meiosis I and II to form ;160 spermatids. Spermatids mature to spermatozoa and become capable of fertilization only after entering the
spermatheca. All subsequent germ cells that initiate meiotic development acquire the female fate. Female germ cell nuclei (presumptive oocytes)
proceed through the pachytene stage of meiotic prophase in the distal arm and progress from diplotene to diakinesis from the loop through the
proximal arm. Moving through the loop, oocytes become more fully enclosed by membrane and grow in size. Distal germ cells are syncytial,
while membrane enclosed diakinesis stage oocytes can remain connected to the syncytium by a narrow rachis (not shown) (White, 1988).
Maturation occurs in the most proximal oocyte in the gonad arm. Ovulation then transports the mature oocyte into the lumen of the
spermatheca where fertilization occurs. The fertilized egg moves from the spermathecal lumen to the uterine lumen through the spermathecal–
uterine valve. Fertilized eggs complete the divisions of meiosis I and II in the uterus. Following maturation and ovulation of the most proximal
oocyte (No. 1), oocyte 2 moves to the most proximal position in the gonad arm and will undergo maturation and ovulation over the course of the
next ;23 min. The tubular gonad arm is covered by 10 somatic sheath cells, the 6 most proximal of which are myoepithelial and capable of
contraction. The lumen of the gonad arm connects proximally to the lumen created by the 24 cells of the somatic spermatheca. The contractile
spermatheca can be considered to be myoepithelial as actin filaments are arrayed in a circumferential lattice (Strome, 1986b), and recently an
unconventional myosin that is expressed in it has been identified (Baker and Titus, 1997; J. Baker and M. Titus, personal communication). Distal
spermathecal cells form a constriction, preventing oocytes from exiting the gonad arm until ovulation when they dilate. Germline development
is reviewed in Kimble and Ward (1988) and Schedl (1997). Somatic structures have been described previously (Hirsh et al., 1976; Kimble and Hirsh,
1979; Strome, 1986b; Creutz et al., 1996; McCarter et al., 1997; Rose et al., 1997).
brief description of these events was first reported in Ward
and Carrel (1979).
Late oogenesis. Late oogenesis occurs primarily during
diakinesis of meiotic prophase I in the proximal gonad arm. At
about 277 min, while the developing oocyte is between the
second and fourth position in the proximal arm, the nucleolus
disappears (Fig. 3a), coinciding with decreased rRNA transcription (Starck et al., 1983). Disassembly of an organized
nucleolus is a common feature of both mitotic and meiotic
divisions (Busch and Smetana, 1970). As the oocyte develops,
the nucleus is usually located in the cell’s distal region, which
is typically the future anterior of the embryo (Goldstein and
Hird, 1996). In most oocytes (83%, n 5 60), the nucleus
showed periods of directed migration where it moved several
micrometers to the oocyte’s distal surface over an ;20-min
period (Fig. 3b), the last migration occurring from approximately 231 to 212 min on average. This off-center placement
of the nucleus is the only sign of polarization in the oocyte,
and its significance is unclear. Oocytes in which the nucleus
fails to reach the distal surface undergo normal embryogenesis
(data not shown), and studies of fertilization suggest that the
sperm entry point is the sole determinant of the anterior–
posterior axis in C. elegans embryos (Goldstein and Hird,
1996). Late oogenesis is also characterized by large increases in
both oocyte nuclear and oocyte cellular volumes. Nuclear
volume increases from ;300 mm 3 at 2140 min to ;700 mm 3
at 210 min, while cellular volume increases from ;12,000 to
;20,000 mm 3. As noted above, cellular volume increase im-
Copyright © 1999 by Academic Press. All rights of reproduction in any form reserved.
115
Oocyte Maturation in C. elegans
FIG. 2. Time line depicting the landmark morphological events of oocyte development, meiotic maturation, ovulation, fertilization, the
meiotic divisions, and early embryogenesis for one oocyte. The end of ovulation (closing of the distal spermatheca) is defined as 0 min and
coincides closely with fertilization. Events from 280 to 180 min are shown. The 26- to 16-min interval is expanded to show the closely coupled
events of oocyte maturation and ovulation. The oocyte arrest point in females occurs after the events of late oogenesis but prior to the events
of maturation and ovulation. However, its exact placement on the time line is arbitrary. See the text for further description, Table 2 for the data
used to construct the time line including standard deviations, Fig. 3 for photographs of events, and Fig. 5a for a sheath activity profile.
mediately prior to maturation is more pronounced in young
adults that have recently switched from spermatogenesis to
oogenesis since oocytes in older adults increase in size earlier
in progression through the proximal gonad arm.
Oocyte meiotic maturation. In adult wild-type hermaphrodites, maturation and ovulation occurs every ;23 min in
each arm and is limited to the most proximal oocyte. Maturation is characterized by two visible events occurring in
quick succession within the oocyte. First, nuclear envelope
breakdown (NEBD) begins at ;26 min as the distinct edge
which separates the nucleus from the cytoplasm begins to
fade (Fig. 3c). By ;2 min postovulation, the nucleus is no
longer recognizable. Second, cortical rearrangement begins at
;23 min, rapidly transforming the oocyte from a cylindrical
to ovoid shape (Fig. 3d). This oocyte shape change appears to
be intrinsically driven and is not dependent on the contractions of the surrounding somatic sheath, since ablation of
sheath cells does not interfere with cortical rearrangement
(McCarter et al., 1997). During meiotic maturation, chromosome arrangement also changes as the bivalents leave diakinesis of meiotic prophase and begin to align on the metaphase
plate (Albertson, 1984; Albertson and Thomson, 1993). A time
course for assembly of the meiosis I spindle has not been
determined.
Oocyte ovulation. Ovulation is characterized by two
processes that occur in the somatic cells: increasing contractile activity in the gonadal sheath cells and relaxation or
dilation of the spermatheca. Prior to maturation and ovulation, the myoepithelial sheath cells of the gonad contract
at a basal level of 10 –13 contractions per minute. Following
oocyte maturation, the rate of sheath contraction increases
to ;19/min (Fig. 5a) and the contractions become increasingly vigorous. At 20.7 min, the distal spermatheca dilates
and is pulled over the most proximal oocyte by the contracting sheath (Fig. 3e) so that by 0 min the oocyte has
entered the lumen of the spermatheca and the distal spermatheca has closed behind the oocyte. Sperm entry at
fertilization is difficult to visualize, but has been observed
to occur immediately upon entry of the oocyte into the
spermatheca (Ward and Carrel, 1979). Cytoplasmic streaming in the oocyte corresponds to the time of fertilization
(but see Table 2, note f). From 13 to 15 min, the
spermathecal-uterine valve dilates (Fig. 3f), and the fertilized egg moves into the uterus.
In the Absence of Sperm, C. elegans Oocytes Are
Capable of Extended Cell Cycle Arrest in
Diakinesis of Meiotic Prophase
In many species, oocytes enter an extended arrest in
meiotic prophase before maturation (Masui and Clarke,
1979; Eppig, 1996). For human oocytes, this arrest can last
for decades. In adult C. elegans hermaphrodites with sperm,
where assembly-line-like production generates a maturing/
ovulating oocyte approximately every 23 min, we believe
that there is no arrest as the signal for maturation (sperm) is
constitutive (see below, and Discussion). However, arrest in
diakinesis is clearly observed when sperm are absent, as in
Copyright © 1999 by Academic Press. All rights of reproduction in any form reserved.
116
McCarter et al.
Copyright © 1999 by Academic Press. All rights of reproduction in any form reserved.
117
Oocyte Maturation in C. elegans
the “females” produced by null mutations in the sex
determination genes fem-1,2,3 and fog-1,2,3 (Schedl, 1997).
In mutant females, where all germ cells develop as oocytes
and no sperm are introduced by mating, oocytes arrest in
diakinesis for hours or even days. Nevertheless, the most
proximal oocyte in female gonad arms do stochastically
mature and ovulate at a very low rate (less than 1/40 the
rate for hermaphrodites), perhaps reflecting an inability of
C. elegans oocytes to maintain arrest indefinitely.
The diakinesis arrest point in females serves as a convenient marker for separating the events of late oogenesis
from those of oocyte maturation and ovulation (Fig. 2).
While maturation is impeded, the events of late oogenesis
(i.e., cellularization, nucleolar disappearance, nuclear migration, cellular and nuclear volume increase, and progression through meiotic prophase up to diakinesis) occur
without interruption in young adult females; arrested oocytes are of full size, lack visible nucleoli, and have large
distally located nuclei with condensed chromosomes in
diakinesis. Because oogenesis continues without oocytes
exiting the gonad, a typical adult female can have 25 or
more oocytes stacked in the gonad arm. While stochastic
maturation results in the first oocyte in the gonad arm
leaving its arrested state after ;14 h, the arrest can last for
days in more distal oocytes. (The 10th oocyte, for example,
can remain in diakinesis for .5 days.)
Spermatozoa, Spermatids, and Spermatocytes
Promote Oocyte Meiotic Maturation
Mating virgin females with wild-type males induces
previously arrested oocytes to begin maturation and ovulation, suggesting that spermatozoa promote oocyte meiotic
maturation. To quantify these observations, we measured
oocyte maturation rates in multiple situations where sperm
were either present or absent. In all cases where wild-type
sperm were present, oocytes matured at an average rate of .2
maturations per gonad arm per hour, including unmated and
mated hermaphrodites, as well as mated females (Figs. 4a and
4b). In all cases where sperm were absent, oocytes matured at
a very low rate of # 0.1, including all unmated females and old
“purged” hermaphrodites which had exhausted their supply of
self-produced sperm (Fig. 4c). The act of mating without
introduction of sperm does not trigger oocyte maturation;
females mated with glp-4(bn2) males raised at 25°C, which
have gonads lacking sperm (Beanan and Strome, 1992), did not
show an increase in the maturation rate of oocytes (Fig. 4c).
Therefore, in all cases, a high rate of oocyte maturation
correlates with the presence of sperm.
During spermatogenesis, primary spermatocytes divide to
form spermatids. Spermatids in turn are activated by contact
with the spermatheca to become motile spermatozoa capable
of fertilization (L’Hernault, 1997). To investigate whether
sperm must be capable of fertilization to promote maturation,
we examined mutants defective in the formation of spermatozoa. At 25°C, fer-1(b232ts) spermatozoa are motility defective with short pseudopods and fer-3(hc3ts) spermatids are
incapable of differentiating into spermatozoa (Argon and
Ward, 1980); neither type of sperm is capable of fertilizing
oocytes. In fer-1(b232ts) and fer-3(hc3ts) hermaphrodites
raised at 25°C, as well as in females mated with fer-1(b232ts)
males raised at 25°C, the rate of oocyte maturation is comparable to the wild-type hermaphrodite rate (Fig. 4d), indicating
that sperm can promote oocyte maturation without being
capable of spermatozoa formation or fertilization. These observations agree with the finding that many sperm-defective
mutants lay oocytes at a rate comparable to wild-type C.
elegans hermaphrodites (L’Hernault et al., 1988; Singson, et
al., 1998).
Interestingly, even developmentally arrested spermatocytes in the gonad arm can induce oocyte maturation. In
spe-4(q347), primary spermatocytes complete meiosis but
fail to undergo cytokinesis to form spermatids (L’Hernault
and Arduengo, 1992). spe-4(q347) young adult hermaphrodites show a rate of oocyte maturation similar to that
observed in wild-type young adult hermaphrodites (1.2 6
0.3 vs 1.3 6 0.3) (Fig. 4d). The resulting ovulations deposit
the spe-4 defective spermatocytes into the uterus where
FIG. 3. Nomarski micrographs of one oocyte in a young adult over 1.5 h of its development. Each row of photographs depicts the
completion of one event (ii, before; iii, during; iv, after). i and v are tracings of ii and iv, respectively, outlining the oocyte’s features before
and after the event. Proximal, lower right corner of each photograph. Ventral surface of worm, upper right corner of each photograph. The
oocyte begins in the second most proximal position in a, ii and b, ii and occupies the most proximal position from a, iii to e, ii. [a, i–v]
Disappearance of nucleolus. The nucleolus (arrow) present in ii has faded from view by iv. [b, i–v] Distal migration of the nucleus. The
nucleus, ;7 mm from the distal surface of the oocyte in ii (arrows), has moved to within ;1 mm by iv. The oocyte surface has become
concave (see text). [c, i–v] Beginning of nuclear envelope breakdown (NEBD). The edge of the nucleus (arrow) which is distinct in ii has faded
from view by iv. The nucleoplasm remains visible until ;2 min after ovulation. [d, i–v] Cortical rearrangement. The oocyte changes from
a cylindrical to ovoid shape (i.e., from a rectangular to elliptical shape in the cross section of the photo). Note the change of the oocyte’s
distal surface (line) from concave in ii to convex in iv. [e, i–v] Ovulation. The distal spermatheca (dotted line) begins constricted and
proximal to the oocyte in ii, dilates and is pulled over the oocyte by the contracting sheath cells in iii, and ends reconstricted and distal
to the oocyte in iv. Fertilization of the oocyte apparently occurs during e, iii to iv, forming the zygote. [f, i–v] The spermatheca– uterine
junction begins constricted and proximal to the zygote in ii, dilates and moves over the zygote in iii, and ends reconstricted and distal to
the zygote in iv. Egg shell formation, which maintains the zygote in the shape established by cortical rearrangement, begins after
fertilization and is completed while the zygote is in the uterus (Ward and Carrel, 1979; Strome, 1986a). Scale bar, 10 mm.
Copyright © 1999 by Academic Press. All rights of reproduction in any form reserved.
118
McCarter et al.
TABLE 2
Time of Events in Oocyte Development, Maturation, and Ovulation
Time of event a
(min)
Developmental stage
Event
Oocyte development
Nucleolus disappears c
Begin last nuclear migration
End last nuclear migration d,e
Begin nuclear envelope breakdown
Begin cortical rearrangement f
Begin entry into spermatheca g
End entry into spermatheca, and fertilization h
Meiotic maturation
Ovulation
Meiotic divisions
Embryogenesis
Begin entry to uterus
End entry to uterus
Meiosis I, polar body 1 i
Meiosis II, polar body 2
Pronuclear fusion
Mitosis I
277 6 28
231 6 9
212 6 6
26 6 2
23 6 2
20.7 6 0.2
0
(by definition)
3 6 1
5 6 1
17 6 3
26 6 4
47 6 13
65 6 17
nb
24
10
10
60
60
60
60
60
60
4
4
7
7
a
Times are rounded up to the nearest minute.
n, the number of oocytes surveyed. Oocytes from both adults and young adults are included. The timing of these events is similar in
both adults and young adults (data not shown).
c
The nucleolus takes 10 –15 min to fade from view. Nucleolar disappearance has been described by electron microscopy (Abi-Rached and
Brun, 1978).
d
In addition to the nucleus moving distally, the distal surface of the oocyte can also bend to meet the nucleus (Fig. 3b), suggesting a
physical connection under tension. The physical movement of the gonad during ovulation of preceding oocytes often disrupts the distal
positioning of nuclei in subsequent oocytes. A single nucleus can be observed to lose its distal position and remigrate distally up to three
times during late oogenesis, each migration occurring between the ovulations of preceding oocytes (data not shown).
e
In most cases (75%, n 5 44) the nucleus is centered along the oocyte’s dorsal–ventral axis (i.e., the sides of the nucleus are 7– 8 mm from
both the dorsal or ventral surface of the oocyte). In the remaining 25% of cases the nucleus is off-center with one side of the nucleus only
2– 6 mm from either the dorsal or ventral surface. Oocytes with nuclei displaced to the dorsal or ventral side showed no defect in maturation,
ovulation, or embryogenesis.
f
Cortical rearrangement has several potential roles. It may allow the first oocyte to separate from the gonadal syncytium, prevent tearing
of the oocyte as it is deformed during ovulation, or serve as the template to maintain the embryo in an ovoid shape until the egg shell is
laid down (Wharton, 1983). Cortical changes at maturation have been investigated in starfish oocytes where they entail formation of actin
spikes extending from the cortex (Otto and Schroeder, 1984), and disassembly of cortical microtubules and intermediate filaments
(Schroeder and Otto, 1991).
g
Spermatids remain in the proximal gonad arm of the hermaphorodite until ovulations begin. The very first ovulation, therefore, differs
from subsequent ovulations in that the oocyte must act as a “plunger” to move a large number of spermatids into the spermatheca.
Subsequent ovulations move smaller numbers of sperm. Despite the larger volume which the first ovulation must move, it occurs equally
rapidly to later ovulations (interval, 0.7 6 0.2 min; n 5 7).
h
During oocyte development (prior to 26 min) and maturation (26 to 0 min), little movement is observed in the oocyte cytoplasm. Upon
spermathecal entry at 0 min, oocyte cytoplasmic granules begin circular streaming. While cytoplasmic streaming was initially thought to
indicate fertilization of the oocyte (Ward and Carrel, 1979; Kimble and Ward, 1988), we observe streaming in ovulated oocytes which are not
fertilized, including ovulated oocytes of fer-1(hc13 and b232) and spe-4(q347) hermaphrodites where sperm are incapable of fertilization, ovulated
oocytes of fog-2(q71) females mated with fer-1(hc13 or b232) males, and rare ovulated oocytes of fem-3(e1996) and fog-3(q443) females where no
sperm are present. Further, streaming is also observed in mature oocytes of the mutants lin-3(n1058) and let-23(sy10) where ovulation is defective
so that the oocyte never reaches the spermatheca (J. McCarter, B. Bartlett, T. Dang, R. J. Hill, M. Lee, and T. Schedl, in preparation, 1998). These
observations indicate that oocyte cytoplasmic streaming requires only oocyte maturation and is not dependent on ovulation or fertilization.
i
Meiosis I and II were timed by the earliest visualization of polar body 1 and polar body 2, respectively (also see Kemphues et al., 1986).
b
they are degraded. In older spe-4(q347) adult hermaphrodites, oocyte maturations are rare, similar to the situation
in females and consistent with the elimination of spe-4
mutant spermatocytes. Taken together, these results indicate that there is a factor from spermatozoa, spermatids,
and spermatocytes (and possibly earlier stages) that can
promote maturation of oocytes in C. elegans.
Sheath Contractile Activity and Spermathecal
Dilation during Ovulation Follow a Reproducible
Sequence Which Requires Maturing Oocytes
Gonadal sheath contractile activity and distal spermathecal dilation, in hermaphrodites, undergo a reproducible
series of changes during ovulation (Fig. 5a). Following
Copyright © 1999 by Academic Press. All rights of reproduction in any form reserved.
119
Oocyte Maturation in C. elegans
FIG. 4. Oocyte maturation rates. Rates were determined by two methods: measuring total embryo production in populations at 20°C (see
Materials and Methods), and by observation of maturation/ovulation with time-lapse microscopy. In a, b, and c, rates from population
samples are graphed whereas in d, rates from microscopy are displayed. Error bars indicate standard deviations. nd, not determined. #
animals, the number of animals surveyed by each method. (a) Wild-type (N2) hermaphrodites. (b) Mated females. Maturation rates are
similar to those observed in adult hermaphrodites. (c) Animals lacking sperm, including unmated females. Oocyte maturations are rare. (d)
Mutants with defective sperm. Rates were determined exclusively by time-lapse microscopy since large numbers of unfertilized oocytes in
populations cannot be counted with accuracy. fer-1(b232ts) and fer-3(hc3ts) animals were raised at 25°C. The fer-1(b232ts) strain contains
him-5(e1490) which has no effect on the maturation rate. The maturation rate observed in unc-24(e138) fem-3(e1996) females mated with
fer-1(b232ts) males is not significantly different from the rate in unc-24(e138) hermaphrodites alone, which is lower than wild-type (data
not shown).
oocyte maturation, the rate of sheath contraction increases
(peaking at ;19/min) and the contractions become more
vigorous (i.e., show increased displacement). As the spermatheca dilates and then reconstricts at ovulation, the
sheath appears to tonically (continuously) contract and
then relax. Its contractile rate drops precipitously (to ;9/
min) and takes several minutes to recover. This cycle is
repeated at the next ovulation. The same pattern is observed in mated females (Fig. 5b) and in mated hermaphrodites (data not shown).
Cyclical changes in sheath activity and spermathecal
relaxation are observed only in the presence of maturing
oocytes. Sheath activity first becomes detectable in
mid-L4 animals, and increases as animals enter adulthood (Fig. 6), yet none of these activity traces show cyclic
changes (data not shown). In all cases where maturing
oocytes are absent, including unmated females (Fig. 5c),
animals with masculinized germlines containing only
spermatids (Fig. 5d), and unmated and mated animals
with tumorous germlines (data not shown), sheath con-
Copyright © 1999 by Academic Press. All rights of reproduction in any form reserved.
120
McCarter et al.
FIG. 5. Sheath contractile activity profiles. Number of sheath contractions per minute is shown over time. Thick line, average. Dotted
line, standard deviation. (a) Wild-type (N2) adult hermaphrodite, one ovulation cycle, n 5 10. Profiles are synchronized at the end of
ovulation (zero min). (b) Mated fog-2(q71) female, one ovulation cycle, n 5 15. Profiles are synchronized at the end of ovulation. (c)
Unmated fog-2(q71) female, n 5 4. No ovulations occurred. Profiles show no dramatic temporal trend and are randomly overlaid. (d)
fem-3(q20gf) hermaphrodites, grown at 25°C, with a masculinized germline (i.e., vast excess of spermatids and no oocytes), n 5 6.
Contractile activity is significantly increased compared to females, but profiles show no dramatic temporal trend and are randomly
overlaid. (e) Unmated females during rare instances of maturation/ovulation, n 5 2. Profiles are synchronized at the end of ovulation. (f)
A composite profile showing the addition of sheath activity from sperm alone (d) and maturing oocytes alone (e) creating a resulting trace
similar to that of an ovulating adult with both sperm and maturing oocytes. Maturation and ovulation was also observed in the
hermaphrodite nematode Caenorhabditis briggsae; sheath activity during the ovulation cycle was similar to that observed in the C. elegans
hermaphrodite (data not shown, n 5 2). Control recordings were also made of anesthetized C. elegans males. Males lack gonadal sheath cells
and contractions like those seen in hermaphrodite gonad arms were not observed (data not shown, n 5 4).
tractile activity traces are relatively constant without
cyclic increases or decreases. Similarly, none of these
conditions cause spermathecal dilation.
Sperm are not required for either sharp increases in sheath
activity or spermathecal dilation. In two rare cases where
oocyte maturation/ovulation was observed in unmated fe-
Copyright © 1999 by Academic Press. All rights of reproduction in any form reserved.
121
Oocyte Maturation in C. elegans
FIG. 6. Average levels of sheath activity in developing wild-type (N2) hermaphrodites. Error bars indicate standard deviations. # animals,
the number of animals of each age surveyed. Total # min, the number of minutes surveyed. Sheath contractions are first observed in mid-L4,
corresponding to our first detection of actin fibers in proximal sheath cells (McCarter et al., 1997) and the onset of spermatogenesis. (Sheath
activity is therefore established before oogenesis begins at the L4/adult molt.) Average levels of sheath activity are higher in young adults
(preovulation) than in ovulating adults because the sheath in young adults contracts at a high steady rate without the trough in activity that
follows each ovulation (Fig. 5a).
males by video microscopy, sheath activity increased and the
spermatheca dilated as the oocyte was ovulated (Fig. 5e).
These findings indicate that the maturing oocyte triggers both
the rise in sheath contractile activity and the dilation of the
spermatheca that occurs at ovulation.
Spermatozoa and Spermatids Promote Sheath
Contractile Activity in the Absence of Oocytes
In unmated females, where neither sperm nor maturing
oocytes are present, sheath activity is ;1.5 contractions/
min, or one-seventh the average hermaphrodite rate (compare Figs. 5a to 5c and 7a to 7d). Mating females with
wild-type males, which introduces sperm and causes oocytes to mature, restores wild-type levels of sheath activity
(Figs. 5b and 7b). Animals with fertilization-defective sperm
also display wild-type levels of sheath activity (Fig. 7c). The
act of mating alone without sperm does not trigger sheath
activity since mating females with glp-4(bn2) males did not
cause increased sheath activity (Fig. 7d). Do sperm cause
the sheath to contract via their effect on oocyte maturation,
or can sperm directly trigger sheath activity independent of
oocytes? Surprisingly, we find that spermatids can stimulate the sheath to contract at 10 –14 contractions/min in the
absence of oocytes. This steady-state rate, similar to that
observed in hermaphrodites between ovulations, is observed in masculinized germ lines, such as fem-3(q20)
(Barton et al., 1987) and gld-1(oz10) (Francis et al., 1995),
which contain only spermatids and no oocytes in the gonad
arm (Figs. 5d and 7e).
We further tested the effect of sperm on sheath contraction in the absence of oocytes by measuring sheath contraction rates in unmated and mated animals with tumorous
germlines. Unmated animals with tumorous gld-1(q485)
fog-3(q443) germlines (Francis et al., 1995; Ellis and
Kimble, 1995) containing neither sperm nor oocytes show
low levels of sheath activity. Introducing spermatozoa by
mating resulted in high levels of sheath activity in all cases
(Fig. 8). Therefore, spermatids present in the gonad arm, as
well as spermatozoa introduced into the spermatheca by
mating, induce sheath activity in the absence of oocytes.
In summary, both sperm and oocytes have effects on the
somatic gonad during ovulation; sperm generate a steadystate rate of sheath activity, while the maturing oocyte both
generates an increase in sheath activity and induces spermathecal relaxation resulting in oocyte ovulation. Interestingly, the effect of sperm and maturing oocytes on sheath
contractile rates appears to be additive. When a trace of
sheath activity from sperm alone (Mog hermaphrodite, Fig.
5d) is added to a trace of sheath activity from maturing
oocytes alone (female, Fig. 5e), the resulting trace (Fig. 5f) is
indistinguishable from that of an ovulating adult with both
sperm and maturating oocytes (Figs. 5a and 5b).
Additional Factors Affect Oocyte Meiotic
Maturation Including Distal–Proximal
Position of the Oocyte
Factors in addition to the presence of sperm play roles in the
regulation of oocyte maturation. In wild-type hermaphrodites,
maturation is never observed to occur out of proximal-todistal order; within the single row of oocytes in the gonad arm
only the most proximal oocyte matures (n 5 60). Because
ablation of the most proximal four sheath cells in wild-type
hermaphrodites delays maturation (McCarter et al., 1997), we
considered the hypothesis that direct contact with proximal
sheath cells (fourth and fifth sheath pair (Kimble and Hirsh,
1979)) might promote maturation. Proximal sheath are connected to oocytes via gap junctions which would allow for
direct cytoplasmic communication (Rose et al., 1997). How-
Copyright © 1999 by Academic Press. All rights of reproduction in any form reserved.
122
McCarter et al.
FIG. 7. Average levels of sheath activity in C. elegans wild-type hermaphrodites and mutants. Error bars indicate standard deviations. #
animals, the number of animals of each genome surveyed. Total # min, the number of minutes surveyed. (a) Wild-type (N2) adult hermaphrodite.
(b) Mated females. (c) Hermaphrodites with defective sperm and females mated to males with defective sperm. The fer-1(b232ts) strain contains
him-5(e1490) which has no effect on sheath activity during ovulation. (d) Unmated females. Both fog-1(q180) and fog-3(q443) female strains
carried unc-13(e51) which has no effect on sheath activity during ovulation (n 5 3). (e) Animals with masculinized germlines. fem-3(q20gf)
animals were maintained at 25°C where their germlines are fully masculinized (Barton et al., 1987). The gld-1(oz10) strain included unc-13(e51).
ever, several observations suggest that direct contact with the
most proximal sheath cells is not required for maturation. In
fem-3(q20) animals raised at 15–20°C excess sperm are produced which fill the proximal gonad arm so that oogenesis is
displaced distally. Oocytes in fem-3(q20) animals with excess
sperm still mature in proximal-to-distal order, but maturation
often begins while the first oocyte is in the loop of the gonad
arm. (The gonad arm loop is enclosed by the second distalmost pair of the five sheath cell pairs in the arm.) We have also
observed oocytes maturing in the loop of the gonad arm in
mutants with the Emo phenotype. In emo-1(oz1) (Iwasaki et
al., 1996), lin-3(n1058), and let-23(sy10) (J. McCarter, B. Bartlett, T. Dang, R. J. Hill, M. Lee, and T. Schedl., in preparation,
1998), where ovulation is defective and mature oocytes are
trapped in the arm, oocytes can mature in proximal-to-distal
order without movement proximally so that oocytes in the
loop eventually mature. Therefore, direct contact with the
most proximal sheath cells is not required for maturation.
DISCUSSION
Our analysis of the temporal coupling of maturation and
ovulation in C. elegans demonstrates that cell cycle progression in one cell (oocyte meiotic maturation) can stimulate neighboring cells to carry out coordinated motor activities (sheath contraction and spermathecal dilation).
Further, regulation of these motor activities likely occurs
without direction from the nervous system since none of
the cells involved are innervated (White et al., 1986). The
evidence presented here suggests a system of regulation
which includes germ cell to germ cell and germ cell to
Copyright © 1999 by Academic Press. All rights of reproduction in any form reserved.
123
Oocyte Maturation in C. elegans
FIG. 8. Average levels of sheath activity in adults of the unc-13(e51)gld-1(q485)fog-3(q443) tumorous strain, unmated and mated to
wild-type males. Filled bars give averages and error bars give standard deviations. (fog-3(q443) was included to eliminate the
spermatogenesis that can occur in q485 in an unc-13 background (Francis et al., 1995).) Data include only animals early in adulthood. In
mated gld-1(q485) animals, sheath activity was steady without peaks and troughs.
somatic cell signaling. First, oocyte maturation is induced
by sperm (i.e., spermatocytes, spermatids, spermatozoa)
temporally and spatially independent of the sperm’s role at
fertilization (Fig. 9a). Second, sperm also promote steadystate sheath contractile activity with or without oocytes
(Fig. 9b). Third, the maturing oocyte modulates sheath
activity during ovulation (Fig. 9c). Fourth, the maturing
oocyte induces spermathecal relaxation during ovulation
(Fig. 9c).
Regulation of Oocyte Meiotic Maturation by Sperm
Stimulation of oocyte production by sperm in C. elegans
has been recognized previously (Nelson et al., 1978; Ward
and Carrel, 1979; L’Hernault et al., 1988; Singson et al.,
1998). Our findings show that oocyte meiotic maturation
(NEBD and cortical rearrangement) is the regulatory point
upon which sperm act. The biochemical mechanism by
which sperm promote oocyte maturation is unknown. Our
studies of oocyte maturation in mutants with defective
spermatogenesis help to establish some of the characteristics of a putative sperm-derived factor promoting oocyte
maturation. First, the factor functions independently of the
interaction between sperm and oocytes during fertilization
since fertilization-defective sperm are still capable of promoting oocyte maturation. Second, the factor is found in
spermatozoa, spermatids, and spe-4 mutant spermatocytes,
indicating that it is produced during spermatogenesis.
Third, the factor may be secreted by sperm and act at a
distance since sperm transferred from males to females and
residing in the spermatheca cause oocyte maturation in the
gonad arm. However, we have not addressed whether sperm
might directly contact the oocyte through the distal spermathecal constriction nor whether the signal might be
indirectly conveyed via the spermatheca or sheath. C.
elegans sperm can be separated from worm carcasses for
biochemical fractionation (Klass and Hirsh, 1981) and can
also be injected into the uterus for in vitro fertilization
(LaMunyon and Ward, 1994). Such techniques may allow
purification of a sperm-produced factor which induces oocyte maturation. In Drosophila, a 36-amino-acid peptide
has been isolated from male seminal fluid which greatly
stimulates ovulation; however, the effect of this peptide on
oocyte maturation, if any, is not known (Chen et al., 1988).
Additional Determinants Affecting Oocyte Meiotic
Maturation
Oocyte maturation is regulated in other ways. First,
maturation occurs only in the most proximal oocyte in the
gonad arm (Fig. 1), even for the rare maturations occurring
in the absence of sperm. This restriction suggests a role for
proximal– distal position in maturation. The somatic gonadal sheath cells promote maturation (McCarter et al.,
1997; Rose et al., 1998). However, direct contact of the
oocyte with the most proximal sheath cells (fourth and fifth
pair, see Kimble and Hirsh, 1979) is not necessary for
maturation; proximal-to-distal maturation occurs normally
in mutants where the most proximal oocyte resides in the
loop region (see Results). One possibility is that the proximal gonad produces a diffusible signal. The most proximal
oocyte would be exposed to a higher concentration of this
signal than more distal oocytes. Signaling within the germline is also likely to be an important part of the restriction
mechanism. The most proximal oocyte is unique in that
there are no oocytes proximally while oocytes in the second
and more distal positions have oocytes both proximally and
distally. The proximal oocyte might produce a signal that
inhibits maturation of distal oocytes. Additionally, the
most proximal oocyte may escape from an inhibitory signal
from distal oocytes. Cytological data have recently supported the view that the most proximal oocyte is distinct
from more distal oocytes; the air-1 protein preferentially
localizes to chromosomes in the most proximal oocyte
while it is primarily distributed in the cytoplasm of more
distal oocytes (J. Schumacher, A. Golden, and P. Donovan,
personal communication).
Second, maturation occurs only in an oocyte which has
completed the events of oocyte development, including
nucleolus breakdown, distal nuclear positioning, meiotic
prophase progression/chromosome condensation, and expansion of oocyte cellular and nuclear volume. Either
dependent pathways or independent but coordinately timed
pathways might ensure that the events of oocyte develop-
Copyright © 1999 by Academic Press. All rights of reproduction in any form reserved.
124
McCarter et al.
FIG. 9. The role of germ cell signaling in maturation and ovulation. (a) Sperm promote oocyte maturation. In the absence of sperm, oocytes
enter prolonged arrest in diakinesis and only rarely mature. (b) Sperm promote sheath contractile activity in the absence of oocytes. This
is true for spermatids in the gonad arm [fem-3(q20gf)] or spermatozoa introduced into the spermatheca of tumorous mutants by mating
[gld-1(q485) hermaphrodite 3 wild-type male]. (c) The maturing oocyte modulates sheath activity and induces spermathecal dilation at
ovulation. Following ovulation, sheath activity decreases to the lowest level in the cycle (see Fig. 5). Possibly, spermathecal reconstriction,
or the zygote in the spermatheca, feeds back on the sheath leading to the observed decrease in activity following ovulation.
ment have been completed before allowing oocyte maturation. The dependence relationships of events within C.
elegans oocyte maturation are currently undefined. For
example, we do not know whether NEBD can occur in the
absence of cortical rearrangement. Experiments suggest
that there are independent pathways for mouse oocyte
maturation where the cdc2/cyclin B complex (MPF) is
needed for NEBD, and Mos and MAP kinase regulate
microtubule and chromatin behavior (Verlhac et al., 1996).
Future studies may allow organization of the events of C.
elegans oogenesis and meiotic maturation into pathways as
has been accomplished for mitotic cell cycle events (Hartwell et al., 1974; Murray, 1992).
Regulation of the Sheath and Spermatheca
Myoepithelial Activities at Ovulation
Ovulation in C. elegans consists of a highly reproducible
series of myoepithelial activities, each change presumably
caused by altered ion channel activity within the cells.
These activities include an increase in sheath contraction
rate and intensity and dilation of the spermatheca leading
to oocyte exit from the gonad arm, followed by a decrease in
sheath contraction rate and intensity and reconstriction of
the spermatheca. Although the sheath and spermatheca
lack innervation (White et al., 1986), a rather complex
motor pattern is still assembled through sperm to sheath
signaling for baseline contractile activity (Fig. 9b) followed
by maturing oocyte to sheath and spermatheca signaling for
contraction and dilation, respectively (Fig. 9c). These nonneuronally mediated contractile activities are similar in
some respects to C. elegans muscular activity for feeding
since pharyngeal pumping can still occur following the
ablation of the entire pharyngeal nervous system (Avery
and Horvitz, 1989).
Cell cycle progression is known to lead to changes in
electrophysiological properties within the cycling cell (Day et
Copyright © 1999 by Academic Press. All rights of reproduction in any form reserved.
125
Oocyte Maturation in C. elegans
FIG. 10. Variations in oocyte meiotic progression between species. The progression of events from late prophase of oogenesis to the
initiation of zygotic development is shown schematically for vertebrates and C. elegans. See text for details. Most vertebrates have meiotic
arrest points in both prophase of meiosis I (oocyte) and at metaphase of meiosis II (unfertilized egg). For vertebrates, meiosis II arrest allows
ovulation to be separated temporally and spatially from fertilization. C. elegans oocytes are capable of arrest in prophase of meiosis I
(females) but in the presence of sperm progress directly to maturation. Since ovulation is immediately followed by fertilization in the
spermatheca, a meiosis II arrest is unnecessary. Vertebrate oocytes arrest in the transcriptionally active diplotene stage while oocytes in C.
elegans females arrest later, in diakinesis. The later arrest in Caenorhabditis elegans may allow a rapid transition to egg production
(maturation, ovulation, and fertilization) following chance mating of a female or “purged” hermaphrodite with a male.
al., 1993). For instance, MPF activity during Xenopus oocyte
maturation can inactivate a delayed rectifier potassium channel (Bruggemann et al., 1997). Ovulation in C. elegans appears
to provide a rare tractable example where cell cycle progression in one cell (the oocyte) leads to changes in contractile
activity of neighboring cells (the sheath and spermatheca),
presumably due to alteration of their electrophysiological
properties. Recent investigations have begun to address the
molecular mechanisms by which maturation and ovulation
are coupled in C. elegans. We have demonstrated a role for the
EGF-like ligand LIN-3 and the receptor tyrosine kinase
LET-23 in oocyte signaling for spermathecal dilation (J. McCarter, B. Bartlett, T. Dang, R. Hill, M. Lee, and T. Schedl, in
preparation). Clandinin and colleagues have identified two
gene products, lfe-1 and lfe-2, that likely act in LET-23 signal
transduction to alter calcium levels within the spermatheca
(Clandinin et al., 1998).
Variations in Meiotic Progression and
Comparative Reproductive Biology
The progression from oocyte to cleaving embryo involves
a similar set of events in most organisms: meiotic matura-
tion, the reductional (MI) and equational (MII) meiotic
divisions, ovulation, and fertilization. However, the order
of events and the position or utilization of control points
can differ (Fig. 10). These differences presumably reflect
adaptations to suit each species’ reproductive biology. C.
elegans is a species with a short life span and a life strategy
based on producing a large number of progeny in as short a
time as possible (Hodgkin and Barnes, 1991). Because of the
worms’ tube-shaped morphology, it produces oocytes in a
single-file assembly-line-like fashion, where only one oocyte within a gonad arm can undergo maturation, ovulation, and fertilization at a time.
For many species, oocytes can undergo a prolonged arrest
in prophase of meiosis I (Masui and Clarke, 1979). For C.
elegans, oocytes arrest in diakinesis of prophase I in the
absence of sperm. In vertebrates, oocyte arrest is relieved in
a periodic manner, seasonally in many amphibians, by the
estrus cycle in many mammals, and by the menstrual cycle
in primates. In contrast, oocytes in C. elegans hermaphrodites with abundant sperm progress through prophase and
mature in an assembly-line-like manner. There is no obvious diakinesis prophase arrest, consistent with a life strategy of producing a large number of progeny in a short time.
Copyright © 1999 by Academic Press. All rights of reproduction in any form reserved.
126
McCarter et al.
In many vertebrates, maturation/ovulation is temporally
and spatially separated from fertilization. Following maturation (NEBD) and MI in the ovary, oocytes are ovulated and
the eggs arrest at a second point, metaphase of MII, until
fertilization. By contrast, for C. elegans, internal fertilization in the spermatheca results in a temporal and spatial
coupling of maturation/ovulation and fertilization. There is
no MII, postovulation, arrest. In the absence of fertilization,
ovulated oocytes fail to complete meiosis, do not form an
egg shell, and are removed from the reproductive tract by
the egg-laying system (Ward and Miwa, 1978).
Selective Advantage in the Regulation
of Maturation by Sperm
Why does a species with self-fertile hermaphrodites like
C. elegans use a sperm-derived factor to stimulate oocyte
maturation? One possibility is that the sperm dependence
of oocyte maturation helps conserve resources following
depletion of self-sperm in anticipation of later mating.
Unmated wild-type hermaphrodites produce an average of
320 progeny, corresponding to the number of self-produced
sperm, whereas mated hermaphrodites can generate as
many as 1400 (Kimble and Ward, 1988). Since males are rare
(Hodgkin et al., 1979), hermaphrodites may travel for days
or a lifetime without mating. Therefore, hermaphrodites
depleted of self-sperm may greatly slow their rate of oocyte
maturation to save valuable oocytes within the gonad arm.
In their conservation of oocytes, hermaphrodites depleted
of self-sperm are behaving like females of related nematode
species like C. remanei which also retain their oocytes
until sperm arrive (our unpublished observations). The
sperm dependence of oocyte maturation may be a mechanism inherited from a male/female ancestor species and
maintained in the hermaphrodite to take advantage of
opportunities for cross-progeny production from mating.
ACKNOWLEDGMENTS
Strains, reagents, experimental protocols, and productive suggestions were provided by Leon Avery, Laura Wilson Berry, Ross
Francis, Bob Goldstein, David Greenstein, Steve L’Hernault, Eric
Lambie, Ann Rose, Bob Waterston, Shelly Weiss, Jim Waddle, and
Sam Ward. We thank Eric Lambie, David Greenstein, and Yen Bui
for their helpful comments. This research was supported by National Science Foundation Grant 9506220 to T.S. During a portion
of this work, T.D. was supported by the National Science Foundation summer undergraduate research program in developmental
biology (BIR-9531558) and B.B. received support from a Howard
Hughes Medical Institute Grant to the Young Scientist Program for
high school student research at Washington University. Some
strains used in this study were provided by the Caenorhabditis
Genetics Center, which is supported by the National Institutes of
Health’s National Center for Research Resources.
REFERENCES
Abi-Rached, M., and Brun, J. L. (1978). Ultrastructural changes in
the nuclear and perinuclear regions of the oogonia and primary
oocytes of C. elegans, Bergerac strain. Rev. Nematol. 1, 63–72.
Albertson, D. G. (1984). Formation of the first cleavage spindle in
nematode embryos. Dev. Biol. 101, 61–72.
Albertson, D. G., and Thomson, J. T. (1993). Segregation of holocentric chromosomes at meiosis in the nematode, Caenorhabditis elegans. Chromosome Res. 1, 15–26.
Argon, Y., and Ward, S. (1980). Caenorhabditis elegans
fertilization-defective mutants with abnormal sperm. Genetics
96, 413– 433.
Avery, L., and Horvitz, H. R. (1989). Pharyngeal pumping continues
after laser killing of the pharyngeal nervous system of C. elegans.
Neuron 3, 473– 485.
Baker, J. P., and Titus, M. A. (1997). A family of unconventional
myosins from the nematode Caenorhabditis elegans. J. Mol.
Biol. 272, 523–535.
Barton, M. K., and Kimble, J. (1990). fog-1, a regulatory gene
required for specification of spermatogenesis in the germ line of
Caenorhabditis elegans. Genetics 125, 29 –39.
Barton, M. K., Schedl, T. B., and Kimble, J. (1987). Gain-of-function
mutations of fem-3, a sex-determination gene in Caenorhabditis
elegans. Genetics 115, 107–119.
Beanan, M. J., and Strome, S. (1992). Characterization of a germ-line
proliferation mutation in Caenorhabditis elegans. Development
116, 755–766.
Bruggemann, A., Stuhmer, W., and Pardo, L. A. (1997). Mitosispromoting factor-mediated suppression of a cloned delayed rectifier potassium channel expressed in Xenopus oocytes. Proc.
Natl. Acad. Sci. USA 94, 537–542.
Busch, H., and Smetana, K. (1970). “The Nucleolus.” Academic
Press, New York.
Chen, P. S., Stumm-Zollinger, E., Aigaki, T., Balmer, J., Bienz, M.,
and Bohlen, P. (1988). A male accessory gland peptide that
regulates reproductive behavior of female D. melanogaster. Cell
54, 291–298.
Clandinin, T. R., DeModena, J. A., and Sternberg, P. W. (1998).
Inositol triphosphate mediates a RAS-independent response to
LET-23 receptor tyrosine kinase activation in C. elegans. Cell 92,
523–533.
Creutz, C. E., Snyder, S. L., Daigle, S. N., and Redick, J. (1996).
Identification, localization, and functional implications of an
abundant nematode annexin. J. Cell Biol. 132, 1079 –1092.
Day, M. L., Pickering, S. J., Johnson, M. H., and Cook, D. I. (1993).
Cell-cycle control of a large-conductance K1 channel in mouse
early embryos. Nature 365, 560 –562.
Downs, S. M. (1995). Ovulation. 2. Control of the resumption of
meiotic maturation in mammalian oocytes. In “Gametes: The
Oocyte” (J. G. Grudzinskas and J. L. Yovich, Eds.), pp. 150 –192.
Cambridge Univ. Press, Cambridge.
Ellis, R. E., and Kimble, J. (1995). The fog-3 gene and regulation of
cell fate in the germ line of Caenorhabditis elegans. Genetics
139, 561–577.
Eppig, J. J. (1993). Regulation of mammalian oocyte maturation. In
“The Ovary” (E. Y. Adashi and P. C. K. Leung, Eds.), pp. 185–208.
Raven Press, New York.
Eppig, J. J. (1996). The ovary: Oogenesis. In “Scientific Essentials of
Reproductive Medicine” (S. G. Hillier, H. C. Kitchener, and J. P.
Neilson, Eds.), pp. 147–159. Saunders, London.
Copyright © 1999 by Academic Press. All rights of reproduction in any form reserved.
127
Oocyte Maturation in C. elegans
Espey, L. L., and Lipner, H. (1994). Ovulation. In “The Physiology
of Reproduction” (E. Knobil and J. D. Neill, Eds.), pp. 725–780.
Raven Press, New York.
Francis, R., Barton, M. K., Kimble, J., and Schedl, T. (1995). gld-1, a
tumor suppressor gene required for oocyte development in Caenorhabditis elegans. Genetics 139, 607– 630.
Goldstein, B., and Hird, S. N. (1996). Specification of the anteroposterior axis in Caenorhabditis elegans. Development 122,
1467–1474.
Hartwell, L. H., Culotti, J., Pringle, J. R., and Reid, B. J. (1974).
Genetic control of the cell division cycle in yeast. Science 183,
46 –51.
Hirsh, D., Oppenheim, D., and Klass, M. (1976). Development of
the reproductive system of Caenorhabditis elegans. Dev. Biol.
49, 200 –219.
Hodgkin, J. (1986). Sex determination in the nematode Caenorhabditis elegans: Analysis of tra-3 suppressors and characterization
of the fem genes. Genetics 114, 15–52.
Hodgkin, J. (1997). Appendix 1: Genetics. In “C. elegans, II” (D. L.
Riddle, T. Blumenthal, B. J. Meyer, and J. R. Priess, Eds.), pp.
881–1048. Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, NY.
Hodgkin, J., and Barnes, T. M. (1991). More is not better: Brood size
and population growth in a self-fertilizing nematode. Proc. R.
Soc. London Ser. B 246, 19 –24.
Hodgkin, J., Edgley, M., Riddle, D., and Albertson, D. (1988).
Genetics. In “The Nematode Caenorhabditis elegans” (W. B.
Wood, Ed.), pp. 491–584. Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, NY.
Hodgkin, J., Horvitz, H. R., and Brenner, S. (1979). Nondisjunction
mutants of the nematode C. elegans. Genetics 91, 67–94.
Iwasaki, K., McCarter, J., Francis, R., and Schedl, T. (1996). emo-1,
a Caenorhabditis elegans Sec61p g homologue, is required for
oocyte development and ovulation. J. Cell Biol. 134, 699 –714.
Kanatani, H., Shirai, H., Nakanishi, K., and Kurokawa, T. (1969).
Isolation and identification of meiosis inducing substance in
starfish Asterias amurensis. Nature 221, 273–274.
Kemphues, K. J., and Strome, S. (1997). Fertilization and establishment of polarity in the embryo. In “C. elegans II” (D. L. Riddle,
T. Blumenthal, B. J. Meyer, and J. R. Priess, Eds.), pp. 335–360.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Kemphues, K. J., Wolf, N., Wood, W. B., and Hirsh, D. (1986). Two
loci required for cytoplasmic organization in early embryos of
Caenorhabditis elegans. Dev. Biol. 113, 449 – 460.
Kimble, J., and Hirsh, D. (1979). Postembryonic cell lineages of the
hermaphrodite and male gonads in Caenorhabditis elegans. Dev.
Biol. 70, 396 – 417.
Kimble, J., and Ward, S. (1988). Germline development and fertilization. In “The Nematode Caenorhabditis elegans” (W. B.
Wood, Ed.), pp. 191–213. Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, NY.
King, R. C. (1970). “Ovarian Development in Drosophila melanogaster.” Academic Press, New York.
Kirby, C., Kusch, M., and Kemphues, K. (1990). Mutations in the
par genes of Caenorhabditis elegans affect cytoplasmic reorganization during the first cell cycle. Dev. Biol. 142, 203–215.
Klass, M. R., and Hirsh, D. (1981). Sperm isolation and biochemical
analysis of the major sperm protein from Caenorhabditis elegans. Dev. Biol. 84, 299 –312.
Kosako, H., Gotoh, Y., and Nishida, E. (1994). Requirement for the
MAP kinase kinase/MAP kinase cascade in Xenopus oocyte
maturation. EMBO J. 13, 2131–2138.
Krantic, S., Dube, F., Quirion, R., and Guerrier, P. (1991). Pharmacology of the Seratonin-induced meiosis reinitiation in Spisula
solidissima oocytes. Dev. Biol. 146, 491– 498.
L’Hernault, S. W. (1997). Spermatogenesis. In “C. elegans, II” (D. L.
Riddle, T. Blumenthal, B. J. Meyer, and J. R. Priess, Eds.), pp.
271–294. Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, NY.
L’Hernault, S. W., and Arduengo, P. M. (1992). Mutation of a
putative sperm membrane protein in Caenorhabditis elegans
prevents sperm differentiation but not its associated meiotic
divisions. J. Cell Biol. 119, 55– 68.
L’Hernault, S. W., Shakes, D. C., and Ward, S. (1988). Developmental genetics of chromosome I spermatogenesis-defective mutants
in the nematode Caenorhabditis elegans. Genetics 120, 435–
452.
LaMunyon, C. W., and Ward, S. (1994). Assessing the viability of
mutant and manipulated sperm by artificial insemination of
Caenorhabditis elegans. Genetics 138, 689 – 692.
Lohka, M. J., Hayes, M. K., and Maller, J. L. (1988). Purification of
maturation-promoting factor, an intracellular regulator of early
mitotic events. Proc. Natl. Acad. Sci. USA 85, 30009 –30013.
Maller, J. L., Gautier, J., Langan, T. A., Lohka, M. J., Shenoy, S.,
Shalloway, D., and Nurse, P. (1989). Maturation-promoting factor and the regulation of the cell cycle. J. Cell Sci. 12, 53– 63.
Masui, Y. (1967). Relative roles of the pituitary, follicle cells, and
progesterone in the induction of oocyte maturation in Rana
pipiens. J. Exp. Zool. 166, 365–376.
Masui, Y., and Clarke, H. J. (1979). Oocyte maturation. Int. Rev.
Cytol. 57, 185–282.
Masui, Y., and C. L. Markert. (1971). Cytoplasmic control of
nuclear behavior during meiotic maturation of frog oocytes. J.
Exp. Zool. 177, 129 –145.
McCarter, J., Bartlett, B., Dang, T., and Schedl, T. (1997). Soma–
germ cell interactions in Caenorhabditis elegans: Multiple
events of hermaphrodite germline development require the somatic sheath and spermathecal lineages. Dev. Biol. 181, 121–143.
Murray, A. W. (1992). Creative blocks: Cell-cycle checkpoints and
feedback controls. Nature 359, 599 – 604.
Myers, C. D., Goh, P. Y., Allen, T. S., Bucher, E. A., and Bogaert, T.
(1996). Developmental genetic analysis of troponin T mutations
in striated and non-striated muscle cells of C. elegans. J. Cell
Biol. 132, 1061–1077.
Nelson, G. A., Lew, K. K., and Ward, S. (1978). Intersex, a
temperature-sensitive mutant of the nematode Caenorhabditis
elegans. Dev. Biol. 66, 386 – 409.
Nurse, P. (1990). Universal control mechanisms regulating onset of
M-phase. Nature 344, 503–508.
Otto, J. J., and Schroeder, T. E. (1984). Assembly– disassembly of
actin bundles in starfish oocytes: An analysis of actin-associated
proteins in the isolated cortex. Dev. Biol. 101, 263–273.
Rose, K. L., Winfrey, V., Hoffman, L., Hall, D. H., Furuta, T., and
Greenstein, D. (1997). The POU-gene ceh-18 promotes gonadal
sheath cell differentiation and function required for meiotic
maturation and ovulation in Caenorhabditis elegans. Dev. Biol.
192, 59 –77.
Sagata, N., Daar, I., Oskarsson, M., Showalter, S. D., and Van de
Woude, G. F. (1989). The product of the mos proto-oncogene as
a candidate initiator for oocyte maturation. Science 245,
643– 646.
Schedl, T. (1997). Developmental genetics of the germ line. In “C.
elegans, II” (D. L. Riddle, T. Blumenthal, B. J. Meyer, and J. R.
Copyright © 1999 by Academic Press. All rights of reproduction in any form reserved.
128
McCarter et al.
Priess, Eds.), pp. 241–269. Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, NY.
Schedl, T., and Kimble, J. (1988). fog-2, a germ-line-specific sex
determination gene required for hermaphrodite spermatogenesis
in Caenorhabditis elegans. Genetics 119, 43– 61.
Schroeder, T. E., and Otto, J. J. (1991). Snoods: A periodic network
containing cytokeratin in the cortex of starfish oocytes. Dev.
Biol. 144, 240 –247.
Singson, A., Mercer, K. B., and L’Hernault, S. W. (1998). The C.
elegans spe-9 gene encodes a sperm transmembrane protein that
contains EGF-like repeats and is required for fertilization. Cell
93, 71–79.
Starck, J., Gibert, M. A., Brun, J., and Bosch, C. (1983). Ribosomal
RNA synthesis and processing during oogenesis of the free living
nematode C. elegans. Comp. Biochem. Physiol. B 75, 575–580.
Strome, S. (1986a). Establishment of asymmetry in early Caenorhabditis elegans embryos: visualization with antibodies to germ
cell components. In “Gametogenesis and the Early Embryo”
(J. G. Gall, Ed.), pp. 77–95. A. R. Liss, New York.
Strome, S. (1986b). Fluorescence visualization of the distribution of
microfilaments in gonads and early embryos of the nematode C.
elegans. J. Cell Biol. 103, 2241–2252.
Sulston, J., and J. Hodgkin. (1988). Methods. In “The Nematode
Caenorhabditis elegans” (W. B. Wood, Ed.), pp. 587– 606. Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Verlhac, M. H., Kubiak, J. Z., Clarke, H. J., and Maro, B. (1994).
Microtubule and chromatin behavior follow MAP kinase activity
but not MPF activity during meiosis in mouse oocytes. Development 120, 1017–1025.
Verlhac, M. H., Kubiak, J. Z., Weber, M., Geraud, G., Colledge,
W. H., Evans, M. J., and Maro, B. (1996). Mos is required for MAP
kinase activation and is involved in microtubule organization
during meiotic maturation in the mouse. Development 122,
815– 822.
Ward, S., and Carrel, J. S. (1979). Fertilization and sperm competition in the nematode Caenorhabditis elegans. Dev. Biol. 73,
304 –321.
Ward, S., and Miwa, J. (1978). Characterization of temperaturesensitive, fertilization-defective mutants of the nematode Caenorhabditis elegans. Genetics 88, 285–303.
Wharton, D. A. (1983). The production and functional morphology
of helminth egg-shells. Parasitology 86, 85–97.
White, J. G. (1988). The anatomy. In “The Nematode Caenorhabditis elegans” (W. B. Wood, Ed.), Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, NY.
White, J. G., Southgate, E., Thomson, J. N., and Brenner, S. (1986).
The structure of the nervous system of Caenorhabditis elegans.
Philos. Trans. R. Soc. London Ser. B Biol. Sci. 314, 1–340.
Wickramasinghe, D., and Albertini, D. F. (1993). Cell cycle control
during mammalian oogenesis. Curr. Top. Dev. Biol. 28, 125–153.
Received for publication July 9, 1998
Accepted August 13, 1998
Copyright © 1999 by Academic Press. All rights of reproduction in any form reserved.